DESCRIPTION(Adapted from the applicant's abstract):Pseudomonas Aeruginosa causes chronic respiratory infections in cystic fibrosis (CF) patients. Such infections are extremely difficult to control since the bacteria exhibit a biofilm-mode of growth rendering P. aeruginosa resistant to antimicrobials and phagocytic cells. Infection of the CF lung is a complex process involving numerous host and bacterial products. Two critical bacterial products required for colonization are flagella and type-IV fimbriae. Initial strains of P. aeruginosa which colonize the CF lung are non-mucoid and highly motile. However, during chronic infections mucoid strains arise and persist and many become nonmotile. The mucoid phenotype is due to the overproduction of the polysaccharide alginate, an important virulence factor protecting the bacteria from host defenses. Flagella, type-IV fimbriae, and alginate are also essential for formation of P. aeruginosa biofilms. Preliminary data indicate that these three key virulence factors are coordinately regulated. The stress-response sigma factor AlgT and the response regulator AlgR represent critical links between these systems. This proposal will focus on understanding the coordinate regulation of these essential virulence determinants. In order to address this, three key questions which constitute the aims of this proposal, must be addressed: (1) By what mechanism does AlgT inhibit flagellin synthesis? This will be addressed by analyzing flagella gene fusions to determine the point in the flagellar gene hierarchy at which AlgT exerts negative control. Genetic approaches will be utilized to identify the direct target of AlgT involved in inhibiting flagellar synthesis. (2) How does the response regulator AlgR control type-IV fimbriae-dependent twitching motility? Genetic and biochemical experiments will be used to determine if signaling through AlgR is required for twitching motility. (3) How are the genes for flagella, type-IV fimbriae, and alginate controlled during biofilm development? Gene fusions in flagellar, type-IV fimbriae, and alginate structural and regulatory genes will be generated and their expression will be monitored temporally and spatially during the process of biofilm formation. Expression levels will be determined in sessile and planktonic growth conditions. These genes will also be differentially regulated to determine if ectopic expression alters the outcome of biofilms. The requirement for signaling through the response regulators AlgR and AlgB will also be addressed. The completion of these aims may translate into therapeutics for biofilm control in CF patients colonized with P. aeruginosa.